This project seeks to define the complexity of cardiomyocyte populations in the adult mouse ventricle, and has a specific emphasis on understanding the nature of proliferative cardiomyocytes that support adult heart regeneration after injury. We begin from the perspective that proliferative cardiomyocytes in the adult mouse heart are mononuclear and diploid, which corresponds to the proliferative population in mouse neonatal and zebrafish adult hearts and to the proliferative population in the embryonic hearts of all species. We propose to overcome several outstanding issues related to this general conceptualization that have impeded progress in the field. First, the extent to which the adult mononuclear diploid cardiomyocyte population is heterogeneous is unknown, particularly as related to subpopulations that have proliferative competence. Second, no molecular markers currently exist that identify proliferative cardiomyocytes from nonproliferative. Third, the dynamics of these populations in the heart over time are completely unknown, but are likely of critical importance in understanding the prevalence of heart failure in the elderly. In Aim 1, we will undertake single cell RNA Seq analysis of mononuclear diploid, mononuclear tetraploid, and binuclear cardiomyocytes isolated from adult mice, and will show that genes or gene isoforms that are differentially expressed define groups and subpopulations of these groups of cardiomyocytes. This approach will demonstrate the extent of cardiomyocyte heterogeneity and at the same time will validate markers of these populations that can be used in a number of subsequent studies. As one particularly important application, we will identify which markers label proliferative cardiomyocytes after injury. In Aim 2, we will address the dynamics of these populations over time, in particular comparing young adult to aged adult mice. We will resolve among several models to explain the observation that cardiomyocyte regeneration declines with advanced age. In all, this project combines an innovative conceptualization with new technology to address questions about the substructure and dynamics of cardiomyocyte populations that have not previously been accessible.